Designed transcription factors with defined target specificity and regulatory function provide invaluable tools for basic and applied research, and for gene therapy. Accordingly, the design of sequence-specific DNA binding domains has been the subject of intense interest for the last two decades. Of the many classes of DNA binding proteins studied, the modular Cys2-His2 zinc finger DNA binding motif has shown the most promise for the production of proteins with tailored DNA binding specificity. The novel architecture of this class of proteins provides for the rapid construction of gene-specific targeting devices. Polydactyl zinc finger proteins are most readily prepared by assembly of modular zinc finger domains recognizing predefined three-nucleotide sequences (See, e.g., Segal, D. J., Dreier, B., Beerli, R. R., and Barbas, C. F., III (1999) Proc. Natl. Acad. Sci. USA 96, 2758-2763; Beerli, R. R., Segal, D. J., Dreier, B., and Barbas, C. F., III (1998) Proc. Natl. Acad. Sci. USA 95, 14628-14633; and Beerli, R. R., Dreier, B., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. USA 97, 1495-1500). Polydactyl proteins can be assembled using variable numbers of zinc finger domains of varied specificity providing DNA binding proteins that not only recognize novel sequences but also sequences of varied length. By combining six zinc finger domains, proteins have been produced that recognize 18 contiguous base pairs of DNA sequence, a DNA address sufficiently complex to specify any locus in the 4 billion-base pair human genome (or any other genome). Fusion of polydactyl zinc finger proteins of this type to activation or repression domains provides transcription factors that efficiently and specifically modulate the expression of both transgenes and endogenous genes (Beerli, R. R., Segal, D. J., Dreier, B., and Barbas, C. F., III (1998) Proc. Natl. Acad. Sci. USA 95, 14628-14633; and Beerli, R. R., Dreier, B., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. USA 97, 1495-1500).
While the availability of designed transcription factors with tailored DNA binding specificities provides novel opportunities in transcriptional regulation, additional applications would be available to ligand-dependent transcription factors. Designer zinc finger proteins dependent on small molecule inducers would have a number of applications, both for the regulation of endogenous genes, and for the development of inducible expression systems for the regulation of transgenes. Natural transcription factors are regulated by a number of different mechanisms, including postranslational modification such as phosphorylation (Janknecht, R., and Hunter, T. (1997) EMBO J 16, 1620-1627; Darnell, J. E., Jr. (1997) Science 277, 1630-1635), or by ligand binding. The prototype ligand-activated transcription factors are members of the nuclear hormone receptor family, including the receptors for sex steroids or adrenocorticoids (Carson-Jurica, M. A., Schrader, W. T., and O'Malley, W. (1990) Endocrine Reviews 11, 201-220; Evans, R. M. (1988) Science 240, 889-895). These receptors are held inactive in the absence of hormone, by association with a number of inactivating factors including hsp90 (Pratt, W. B., and Toft, D. O. (1997) Endocrine Rev. 18, 306-360). Upon ligand binding, nuclear hormone receptors dissociate from the inactivating complex, dimerize, and become able to bind DNA and activate transcription (Carson-Jurica, M. A., Schrader, W. T., and O'Malley, W. (1990) Endocrine Reviews 11, 201-220; Evans, R. M. (1988) Science 240, 889-89512-14; and Pratt, W. B., and Toft, D. O. (1997) Endocrine Rev. 18, 306-360). Significantly, not only hormone binding but also inactivation and dimerization functions reside within the ligand binding domain (LBD) of these proteins (Beato, M. (1989) Cell 56, 335-344). This fact has been exploited experimentally and steroid hormone receptor LBDs have found wide use as tools to render heterologous proteins hormone-dependent.
In particular, the estrogen receptor (ER) LBD has been used to render the functions of c-Myc (Eilers, M., Picard, D., Yamamoto, K. R., and Bishop, J. M. (1989) Nature 340, 66-68), c-Fos (Superti-Furga, G., Bergers, G., Picard, D., and Busslinger, M. (1991) Proc. Natl. Acad. Sci. USA 88, 5114-5118), and even the cytoplasmic kinase c-Raf (. Samuels, M. L., Weber, M. J., Bishop, J. M., and McMahon, M. (1993) Mol. Cell. Biol. 13, 6241-6252) hormone-dependent. To develop an inducible expression system for use in basic research and gene therapy, the availability of ligand-dependent transcriptional regulators is a prerequisite. Preferentially, these regulators would be activated by a small molecule inducer with no other biological activity, bind specific sequences present only in the target promoter, and have low immunogenicity. A number of ligand-regulated artificial transcription factors have been generated by various means, using functional domains derived from either prokaryotes (Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551 20. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. (1995) Science 268, 1766-1769 21. Labow, M. A., Baim, S. B., Shenk, T., and Levine, A. J. (1990) Mol. Cell. Biol. 10, 3343-3356 22. Baim, S. B., Labow, M. A., Levine, A. J., and Shenk, T. (1991) Proc. Natl. Acad. Sci. USA 88, 5072-5076) or eukaryotes (Christopherson, K. S., Mark, M. R., Bajaj, V., and Godowski, P. J. (1992) Proc. Natl. Acad. Sci. USA 89, 6314-6318 24. No, D., Yao, T.-P., and Evans, R. M. (1996) Proc. Natl. Acad. Sci. USA 93, 3346-3351 25. Wang, Y., O'Malley, B. W., Jr., Tsai, S., and O'Malley, B. W. (1994) Proc. Natl. Acad. Sci. USA 91, 8180-8184 Beerli et al. -35-26. Wang, Y., Xu, J., Pierson, T., O'Malley, B. W., and Tsai, S. Y. (1997) Gene Therapy 4, 432-441 27. Braselmann, S., Graninger, P., and Busslinger, M. (1993) Proc. Natl. Acad Sci. USA 90, 1657-1661 28. Louvion, J. F., Havaux-Copf, B., and Picard, D. (1993) Gene 131, 129-134 29. Rivera, V. M., Clackson, T., Natesan, S., Pollock, R., Amara, J. F., Keenan, T., Magari, S. R., Phillips, T., Courage, N. L., Cerasoli, F., Jr., Holt, D. A., and Gilman, M. (1996) Nature. Med. 2, 1028-1032).
Of the functional domains derived from eukaryotic proteins, nuclear hormone receptor LBDs have been the most widely used. In particular, regulators based on the Gal4 DNA binding domain (DBD) fused to a human ER (Braselmann, S., Graninger, P., and Busslinger, M. (1993) Proc. Natl. Acad. Sci. USA 90, 1657-1661; Louvion, J. F., Havaux-Copf, B., and Picard, D. (1993) Gene 131, 129-134) or progesterone receptor (PR) LBD; (Wang, Y., O'Malley, B. W., Jr., Tsai, S., and O'Malley, B. W. (1994) Proc. Natl. Acad. Sci. USA 91, 8180-8184; Wang, Y., Xu, J., Pierson, T., O'Malley, B. W., and Tsai, S. Y. (1997) Gene Therapy 4, 432-441), as well as the ecdysone-inducible system based on the Drosophila ecdysone receptor-(EcR) and the mammalian retinoid X receptor (RXR) (Christopherson, K. S., Mark, M. R., Bajaj, V., and Godowski, P. J. (1992) Proc. Natl. Acad. Sci. USA 89, 6314-6318; No, D., Yao, T.-P., and Evans, R. M. (1996) Proc. Natl. Acad. Sci. USA 93, 3346-3351) have been described. Compared to the heterodimeric EcR/RXR system, regulators based on the ER and PR LBDs have the important advantage that they function as homodimers and require the delivery of only one cDNA. However, while ecdysone has no known biological effect on mammalian cells, estrogen and progesterone will elicit a biological response in cells or tissues that express the endogenous steroid receptors. With the availability of a mutated ER and a truncated PR LBDs that have lost responsiveness to their natural ligands but not to synthetic antagonists such as 4-hydroxytamoxifen (4-OHT) (Littlewood, T. D., Hancock, D. C., Danielian, P. S., Parker, M. G., and Evan, G. I. (1995) Nucl. Acids Res. 23, 1686-1690) or RU486 (Vegeto, E., Allan, G. F., Schrader, W. T., Tsai, M.-J., McDonnell, D. P., and O'Malley, B. W. (1992) Cell 69, 703-713), respectively, this is no longer of great concern. Thus, steroid hormone receptor LBD-based inducible expression systems can be developed that function independently of the endogenous steroid receptors. To date, this has been shown for the PR LBD through the development of an RU486-inducible expression system based on the Gal4 DBD (Wang, Y., O'Malley, B. W., Jr., Tsai, S., and O'Malley, B. W. (1994) Proc. Natl. Acad. Sci. USA 91, 8180-8184; Wang, Y., Xu, J., Pierson, T., O'Malley, B. W., and Tsai, S. Y. (1997) Gene Therapy 4, 432-441). An inducible expression system based on a point-mutated (G525R) ER LBD (Littlewood, T. D., Hancock, D. C., Danielian, P. S., Parker, M. G., and Evan, G. I. (1995) Nucl. Acids Res. 23, 1686-1690) that has lost the responsiveness to estrogen but not the antagonist 4-OHT has not been described to date. Designed zinc finger proteins have a number of advantages as compared to other DBDs, including the one derived from Gal4, since the ability to engineer DNA binding specificities allows ligand-dependent regulators to be directed to any desired artificial or natural promoter. Here we explore the utility of fusion proteins between designed zinc finger proteins and nuclear hormone receptor LBDs for the inducible control of gene expression.